Proteins destined for transport into or across cell membranes are usually translated with a signal sequence that directs the newly synthesized protein to the appropriate membrane translocation system. The primary structure of signal sequences is highly variable among different proteins. Signal sequences that target proteins for export from the cytosol generally contain a short stretch (7-20 residues) of hydrophobic amino acids. In most cases, the signal sequence is located at the amino terminus of a nascent protein and is proteolytically removed on the trans side of the membrane (e.g. lumen of endoplasmic reticulum, bacterial periplasm, intercisternal space of mitochondria and chloroplasts), although examples of mature proteins containing uncleaved or internal signal sequences have been described. Export signal sequences may be interchanged among different proteins, even proteins of different species of organisms.
Many secreted eucaryotic proteins interact with target cells to bring about physiological responses such as growth, differentiation and/or activation. These activities make secreted proteins biologically interesting molecules which are potentially valuable as therapeutics or as targets for ligands. Of the estimated 60,000 to 100,000 human genes, about 25% carry a signal peptide and only about 4% are secreted extracellularly. Clearly, approaches which allow rapid and accurate identification of secreted proteins are important tools for gene-based drug discovery programs.
With advances in techniques for sequencing cDNAs, many expressed sequence tags (ESTs) have been generated which have enhanced the process of identifying novel secreted proteins as compared to the conventional reverse genetics approaches. However, EST's are small random cDNA sequences and thus it becomes hard to identify secretion signal sequence that is normally present in the 5' end of cDNA encoding secreted protein. Moreover, after an EST carrying a potential secretion signal sequence is identified based on the homology search, it has to be authenticated in a functional assay. Thus a screen based on selection of functional secretion signals from random cDNA libraries would greatly simplify the process of obtaining novel secreted genes.
Secretion signal trap is one such method to clone 5' ends of cDNAs encoding for secreted proteins from a random cDNA library. Generally, signal trapping relies on secretion of a reporter polypeptide by signal sequences present in a cDNA library. The secreted reporter polypeptide may be detected by a variety of assays based upon growth selection, enzymatic activity or immune reactivity. Examples of signal trap cloning procedures include the following.
U.S. Pat. No. 5,536,637 and Klein et al. Proc. Natl. Acad. Sci. USA 93, 7108-7113 (1996) describe signal trap cloning in yeast using the yeast invertase polypeptide as a reporter.
Imai et al. J. Biol. Chem. 271, 21514-21521 (1996) describe signal trap cloning in mammalian cells using CD4 as a reporter and identifying signal sequences by screening for surface expression of CD4 antigen.
U.S. Pat No. 5,525,486, Shirozu et al. Genomics 37, 273-280 (1996) and Tashiro et al. Science 261, 600-603 (1993) describe signal trap cloning in mammalian cells and identify signal sequences by screening for surface expression of IL-2 receptor fusion proteins.
U.S. Pat. No. 5,037,760 describes signal trap cloning in Bacillus using .alpha.-amylase and .beta.-lactamase as reporter genes.
Published PCT Application No. WO96/40904 describes signal trap cloning by selection for growth of factor-dependent cell lines and screening with tagging reagents for surface expression of growth factor receptors.
Previous approaches to identifying mammalian secreted and transmembrane protein by signal trapping in yeast and prokaryotic systems have a disadvantage in that the machinery that translocates proteins across the membrane of the endoplasmic reticulum (ER) and the mechanisms that process proteins in the ER-golgi are different in mammalian cells. For example, Saccromyces cerivisiae utilizes both a cotranslational and posttranslational mechanism to transport proteins containing signal sequences and mutants in the yeast SRP54 protein, which is integral to the cotranslational mechanism, are viable. Mammalian cells appear to have evolved a special dependence on the cotranslational mechanism and posttranslational modifications are more complex. Thus, many mammalian secreted or transmembrane proteins constructs may not express or sort properly in yeast and, conversely, many mammalian protein sequences appear to function aberrantly as signal sequences.
The above approaches to signal trapping in mammalian cells also lack a convenient selection method for signal sequences in mammalian host cells. Methods described to date involve screening many clones either by enzyme activity or immunoassay for secretion with no efficient way to select against clones not containing functional signal sequences. It would be desirable to provide positive selection for secretion from mammalian cells and reduce the need to screen all clones for signal sequences.
Accordingly, it is an object of the invention to provide signal trap vectors and related methods and compositions for rapidly and accurately identifying novel secreted proteins in mammalian host cells.